For detailed studies of the physiological functions of reactive sulfur species (RSS) such as H₂S and sulfane sulfur, we set out to develop a highly selective and easy-to-use fluorescence probe for H₂S. We designed and synthesized a novel fluorescence probe for H₂S, HSip-1 (Hydrogen Sulfide imaging probe-1), utilizing macroazacyclic complex chemistry with copper ion (II) (J. Am. Chem. Soc. 133, 18003-18005 (2011)). HSip-1 showed the fluorescence increase (by 50 fold) within several seconds upon addition of 10 μM H₂S, whereas almost no fluorescence increment was observed upon addition of 10 mM GSH. HSip-1 also showed high selectivity over other biothiols, ROS, and RNS. We could also visualize H₂S in HeLa cells with HSip-1 DA (a cell-membrane permeable derivative of HSip-1) upon addition of Na₂S. Moreover, we applied HSip-1 to the detection of enzymatic activity of H₂S-producing enzymes in vitro. We successfully monitored the time-dependent H₂S production by 3-mercaptopyruvate sulfurtransferase (3MST) and cystathionine γ-lyase (CSE). We then applied HSip-1 to the inhibitor high-throughput screening (HTS) of a chemical library containing 170,000 compounds from The University of Tokyo, Drug Discovery Initiative, and found selective inhibitors for 3MST and CSE (Sci. Rep. 7:40227 (2017)). We also recently developed a fluorescence probe for sulfane sulfur (Chem. Commun., 53, 1064-1067 (2017)).